Corn root preferential promoters and uses thereof

ABSTRACT

The invention relates to the isolation of promoters from corn capable of directing transcription of an operably linked foreign DNA sequence preferentially, selectively or exclusively in the roots of plants, such as corn plants. The invention also relates to the use of chimeric genes for the preferential or selective expression of biologically active RNA of interest in the roots of plants, such as corn plants. Plants, such as corn plants, comprising corn root preferential or selective promoters operably linked to a foreign DNA sequence which, upon transcription, yield biologically active RNA preferentially or selectively in the roots of plants are also provided.

FIELD OF THE INVENTION

[0001] The invention relates to the isolation of promoters from corncapable of directing transcription of an operably linked foreign DNAsequence preferentially, selectively or exclusively in the roots ofplants, such as corn plants. The invention also relates to the use ofchimeric genes for the preferential or selective expression ofbiologically active RNA of interest in the roots of plants, such as cornplants. Plants, such as corn plants, comprising corn root preferentialor selective promoters operably linked to a foreign DNA sequence which,upon transcription, yield biologically active RNA preferentially orselectively in the roots of plants are also provided.

DESCRIPTION OF RELATED ART

[0002] A significant consideration in the production of transgenicplants is obtaining sufficient levels of expression of the transgene inthe tissues of interest in a preferential or selective way. In this way,potential drawbacks associated with the constitutive expression of thetranscript, such as yield drag, may be avoided. Selection of appropriatepromoters is crucial for obtaining the pattern of expression of interestwith a particular transgene.

[0003] Selective expression of transgenes in roots of plants,particularly cereal plants, such as corn, is considered to bepotentially commercially important, e.g. for alteration of the functionof root tissue, resistance to pathogens or pests with a preference forattack of roots (such as nematodes, corn rootworm etc.), resistance toherbicides or adverse environmental conditions (such as drought or soilcomposition).

[0004] U.S. Pat. No. 5,633,363 describes a 4.7 kb upstream promoterregion designated ZRP2 isolated from maize and attributes a particularutility to this promoter region in driving root preferential expressionof heterologous genes.

[0005] WO 97/44448 relates generally to mechanisms of gene expression inplants and more specifically to regulation of expression of genes inplants in a tissue-specific manner particularly in roots. A method forisolation of transcriptional regulatory elements that contribute totissue-preferred gene expression is disclosed.

[0006] WO 00/15662 describes a promoter of a glycine rich protein(zmGRP3) whose transcripts accumulate exclusively in roots of youngmaize seedlings following developmentally specific patterns.

[0007] WO 00/070068 and WO 00/70066 describe respectively the maize RS81and RS324 promoters which are promoters of genes expressed in maize roottissue but not in kernel tissue and in molecular analysis were describedto have a root-specific expression profile.

[0008] Despite the fact that corn root preferential promoters areavailable in the art, a need remains for alternative promoters capableof preferential or selective root selective expression, e.g. for theindependent expression of several foreign DNA sequences of interestwithout the possibility of post-transcriptional silencing due to the useof the same promoter. In addition, the known corn root preferentialpromoters, each direct a particular temporal, spatial and/ordevelopmental expression pattern, which does not always suit particulargoals. There remains thus a need for novel corn root preferentialpromoters with the capacity to control transcription in roots,preferably in a more selective manner, and also preferably resulting ina highly abundant transcription product.

SUMMARY AND OBJECTS OF THE INVENTION

[0009] It is an object of the invention to provide corn rootpreferential promoters comprising a nucleotide sequence selected fromthe following group of nucleotide sequences:

[0010] a. a nucleotide sequence comprising the nucleotide sequence ofSEQ ID No 1 from the nucleotide at position 1 to the nucleotide atposition 338. or the nucleotide sequence of SEQ ID No 2 from thenucleotide sequence at position 11 to the nucleotide at position 1196(“GL4 promoter”;

[0011] b. a nucleotide sequence comprising the nucleotide sequence ofSEQ ID No 14 from the nucleotide at position 1 to the nucleotide atposition 1280 (“GL5 promoter”);

[0012] c. a nucleotide sequence comprising the nucleotide sequence ofabout an 400 bp to an about 1300 bp DNA fragment from the 5′ end of acorn root preferential gene encoding a mRNA, from which a cDNA can beprepared that comprises the complement of the nucleotide sequence of SEQID No 7 or SEQ ID No 8 or SEQ ID No 9 or SEQ ID No 10;

[0013] d. a nucleotide sequence comprising the nucleotide sequence of anabout 400 bp to an about 1300 bp DNA fragment from the 5′ end of a cornroot preferential gene encoding a mRNA from which a cDNA can be preparedthat contains a nucleotide sequence encoding a polypeptide with theamino acid of SEQ ID No 4 or 6;

[0014] e. a nucleotide sequence comprising the nucleotide sequence of anabout 400 bp to an about 1300 bp DNA fragment from the 5′ end of a cornroot preferential gene encoding a mRNA, from which a cDNA can beprepared that comprises a nucleotide sequence having at least 75%, atleast 80%, at least 90%, at least 95%, or is identical to the nucleotidesequence of any of SEQ ID No 3, 5 or 11;

[0015] f. a nucleotide sequence comprising the nucleotide sequencehaving at least 70% at least 80%, at least 90%, at least 95%, or isidentical to any of said nucleotide sequence mentioned under a), b),c),d), e), or f) ;or

[0016] g. a nucleotide sequence comprising the nucleotide sequence of anabout 400 bp to an about 1300 bp DNA fragment hybridizing understringent conditions with a DNA fragment having said nucleotide sequencementioned under a), b), c), d), e) or f).

[0017] The corn root preferential promoters may be comprised within acorn root preferential promoter region, and may further comprise thenucleotide sequence of SEQ ID 1 from the nucleotide at position 339 tothe nucleotide at position 366 or the nucleotide sequence of SEQ ID 14from the nucleotide at position 1281 to the nucleotide at position 1308.

[0018] It is another object of the invention to provide chimeric genescomprising the following operably linked DNA regions: a corn rootpreferential promoter according to the invention; a heterologous DNAregion encoding a biologically active RNA of interest; and atranscription termination and polyadenylation signal active in plantcells. The biologically active RNA may encode a protein of interest,such as a protein which when expressed in the cells of a plant conferspest or pathogen resistance to said plant. The biologically active RNAmay also be an antisense, sense or double stranded RNA useful forpost-transcriptional silencing of a target gene of interest.

[0019] Also provided are plant cells and plants or seeds thereof,particularly cereal plants, such as corn plants comprising a chimericgene according to the invention.

[0020] It is yet another objective to provide a method for expressing abiologically active RNA preferentially in the roots of a plant, such asa corn plant, comprising the steps of: providing the cells of the rootsof said plants with a chimeric gene according to the invention; andgrowing the plants.

[0021] The invention further provides the use of a corn rootpreferential promoter according to the invention for preferentialexpression of a biologically active RNA in roots of a plant, such as acorn plant.

[0022] It is yet another object of the invention to provide isolated DNAmolecules comprising a nucleotide sequence encoding a protein comprisingthe amino acid sequence of SEQ ID No 4 or SEQ ID No 6, particularly anucleotide sequence selected from the group of SEQ ID No 3; SEQ ID No 5and SEQ ID No 11 and the use thereof for isolation of a corn rootpreferential promoter or promoter region.

[0023] In yet another embodiment the invention provides a method forisolating a corn root preferential promoter region, comprising the stepsof: identifying a genomic fragment encoding an RNA transcript from whicha cDNA can be synthesized, which cDNA comprises the nucleotide sequenceof SEQ ID 3 or SEQ ID No 5 or functional equivalents; and isolating aDNA region upstream of a nucleotide sequence encoding the protein withthe amino acid of SEQ ID No 4 or SEQ ID No 6 or functional equivalents.Also provided are corn root preferential promoters obtained by thismethod.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1: Alignment of the nucleotide sequences for cDNAs GL4, GL5and GL12. Gaps in the sequence introduced for optimal alignment areindicated by a dash.

[0025]FIG. 2: Nucleotide sequence for the short corn root preferentialpromoter region from GL4.

[0026]FIG. 3: Nucleotide sequence for the long corn root preferentialpromoter region from GL4.

[0027]FIG. 4: Nucleotide sequence for the corn root preferentialpromoter region from GL5.

[0028]FIG. 5: Schematic representation of pTWV011. LB: left T-DNAborder; 3′nos: 3′ end of the nopaline synthase gene; bar: bialaphosresistance coding region; P35S3: promoter region of the 35S transcriptof CaMV; 3′ 35S: 3′ end of the 35S transcript of CaMV; isp1a: codingregion for insecticidal secreted protein 1a from Brevibacilluslaterosporus; 5′gl4: leader region of the GL4 promoter region; Pgl4:corn root selective promoter GL4; isp2a: coding region for insecticidalsecreted protein 2a from Brevibacillus laterosporus; RB: right T-DNAborder region; nptl homology: region of homology with helperTi-plasmids; ORI colE1: colE1 origin of replication; ORI pVS1: origin ofreplication for Pseudomonas; PaadA: bacterial promoter of theaminoglycoside adenyltransferase conferring resistance to streptomycinand spectinomycin; aadA: coding region of the aminoglycosideadenyltransferase gene; 3′ aadA: 3′ end of the aminoglycosideadenyltransferase gene.

[0029]FIG. 6: Schematic representation of pTWV018. LB: left T-DNAborder; 3′nos: 3′ end of the nopaline synthase gene; bar: bialaphosresistance coding region; P35S3: promoter region of the 35S transcriptof CaMV; 3′ 35S: 3′ end of the 35S transcript of CaMV; isp1a: codingregion for insecticidal secreted protein 1a from Brevibacilluslaterosporus; 5′gl5: leader region of the GL5 promoter region; Pgl5:corn root selective promoter GL5; isp2a: coding region for insecticidalsecreted protein 2a from Brevibacillus laterosporus; RB: right T-DNAborder region; nptl homology: region of homology with helperTi-plasmids; ORI colE 1: colE1 origin of replication; ORI pVS1: originof replication for Pseudomonas; PaadA: bacterial promoter of theaminoglycoside adenyltransferase conferring resistance to streptomycinand spectinomycin; aadA: coding region of the aminoglycosideadenyltransferase gene; 3′ aadA: 3′ end of the aminoglycosideadenyltransferase gene.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0030] The invention is based on the finding that the promotersdescribed herein are particularly suited for the preferential andabundant expression (i.e. transcription or transcription andtranslation) of an operably linked foreign DNA in roots of plants,particularly cereal plants such as corn.

[0031] In one embodiment of the invention, a corn root preferentialpromoter region is provided comprising the nucleotide sequence of SEQ ID1 of about 400 bp. In another embodiment, a corn root preferentialpromoter region is provided comprising the nucleotide sequence of SEQ IDNo 2 of about 1200 bp. In yet another embodiment, a corn rootpreferential promoter region is provided comprising the nucleotidesequence of SEQ ID No 14 from the nucleotide at position 1 to thenucleotide at position 1280.

[0032] As used herein “corn” refers to maize i.e. Zea mays L.

[0033] As used herein, the term “promoter” denotes any DNA which isrecognized and bound (directly or indirectly) by a DNA-dependentRNA-polymerase during initiation of transcription. A promoter includesthe transcription initiation site, and binding sites for transcriptioninitiation factors and RNA polymerase, and can comprise various othersites (e.g., enhancers), at which gene expression regulatory proteinsmay bind.

[0034] The term “regulatory region”, as used herein, means any DNA, thatis involved in driving transcription and controlling (i.e., regulating)the timing and level of transcription of a given DNA sequence, such as aDNA coding for a protein or polypeptide. For example, a 5′ regulatoryregion (or “promoter region”) is a DNA sequence located upstream (i.e.,5′) of a coding sequence and which comprises the promoter and the5′-untranslated leader sequence. A 3′ regulatory region is a DNAsequence located downstream (i.e., 3′) of the coding sequence and whichcomprises suitable transcription 3′ end formation (and/or regulation)signals, including one or more polyadenylation signals.

[0035] The term “gene” means any DNA fragment comprising a DNA region(the “transcribed DNA region”) that is transcribed into a RNA molecule(e.g., a mRNA) in a cell under control of suitable regulatory regions,e.g., a plant expressible promoter region. A gene may thus compriseseveral operably linked DNA fragments such as a promoter, a 5′untranslated leader sequence, a coding region, and a 3′ untranslatedregion comprising a polyadenylation site. An endogenous plant gene is agene which is naturally found in a plant species. A chimeric gene is anygene which is not normally found in a plant species or, alternatively,any gene in which the promoter is not associated in nature with part orall of the transcribed DNA region or with at least one other regulatoryregions of the gene.

[0036] The term “expression of a gene” refers to the process wherein aDNA region under control of regulatory regions, particularly thepromoter, is transcribed into an RNA which is biologically active, i.e.,which is either capable of interaction with another nucleic acid orwhich is capable of being translated into a biologically activepolypeptide or protein. A gene is said to encode an RNA when the endproduct of the expression of the gene is biologically active RNA, suchas an antisense RNA or a ribozyme. A gene is said to encode a proteinwhen the end product of the expression of the gene is a biologicallyactive protein or polypeptide.

[0037] The term “root-selective”, with respect to the expression of aDNA in accordance with this invention, refers to, for practicalpurposes, the highly specific, expression of a DNA in cells of roots ofplants, such as corn plants (“corn-root-selective”). In other words,transcript levels of a DNA in tissues different of root plants is eitherbelow detection or very low (less than about 0.2 picograms per microgramtotal RNA).

[0038] The term “root-preferential” with respect to the expression of aDNA in accordance with this invention, refers to an expression patternwhereby the DNA is expressed predominantly in roots, but expression canbe identified in other tissues of the plant. In one embodiment of thepresent invention, expression in roots may be enhanced by about 2 toabout 10 times higher in roots than in other tissues.

[0039] It will be clear that having read these embodiments, the personskilled in the art can easily identify and use functional equivalentpromoters for the same purposes.

[0040] DNA sequences which have a promoter activity substantiallysimilar to the corn root preferential promoters comprising thenucleotide sequence of SEQ ID 1 from the nucleotide at position 1 to thenucleotide at position 338 or SEQ ID 2 from the nucleotide at position11 to the nucleotide at position 1196 or SEQ ID 14 from the nucleotideat position 1 to the nucleotide.at position 1280, or parts thereofhaving promoter activity, are functional equivalents of these promoters.These functional equivalent promoters may hybridize with the corn rootpreferential promoter regions comprising the nucleotide sequence of SEQID 1 or of SEQ ID No 2 or of SEQ ID 14 under stringent hybridizationconditions.

[0041] “Stringent hybridization conditions” as used herein means thathybridization will generally occur if there is at least 95% or even atleast 97% sequence identity between the probe and the target sequence.Examples of stringent hybridization conditions are overnight incubationin a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrierDNA such as salmon sperm DNA, followed by washing the hybridizationsupport in 0.1×SSC at approximately 65° C. Other hybridization and washconditions are well known and are exemplified in Sambrook et al,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), particularly chapter 11.

[0042] Other functional equivalent promoters comprise nucleotidesequences which can be amplified using oligonucleotide primerscomprising at least about 25, at least about 50, or at least about 100consecutive nucleotides selected from the nucleotide sequence of SEQ ID1 or SEQ ID 2, in a polymerase chain amplification reaction. Examples ofsuch oligonucleotide primers are GVK 29 (SEQ ID No 9) and GVK 30 (SEQ IDNo 10).

[0043] Functionally equivalent promoters may be isolated e.g. fromdifferent corn varieties. They may also be made by modifying isolatedcorn root-preferential promoters through addition, substitution,deletion or insertion of nucleotides. They can also be completely orpartly synthesized.

[0044] Alternatively, functional equivalent promoters may be isolatedusing a cDNA of a transcript which is expressed at a high level in rootsof a plant, such as a corn plant, as a probe to isolate the genomic DNAupstream of the nucleotide sequence corresponding to the nucleotidesequence of the cDNA. As used herein “cDNA” is used to indicate both thefirst strand cDNA (complementary to the mRNA) as well as the strandcomplementary thereto (and thus identical to the mRNA except that U isreplaced by T) or a double stranded cDNA fragment. In accordance withthis invention, corn root selective cDNAs and their corresponding plantgenomic DNA fragments may be identified as follows:

[0045] a cDNA library may be constructed starting from mRNA isolatedfrom roots and the cDNA library subjected to differential screening inorder to identify an mRNA which is preferentially present in roots whencompared to other plant tissues including but not limited to: leaves,seeds, stems, reproductive organs, and the like. Alternatively, the cDNAlibrary may screened with oligonucleotides, that are deduced from adetermined amino acid sequence of an isolated protein, that has beenidentified to be preferentially present in the roots. Furthermore, it ispossible to use the same oligonucleotides in a nested-PCR approach andto use the amplified fragment(s) as a probe to screen the library. Thecorn root preferential cDNA library can be constructed from a pool ofmRNAs, isolated at different stages of corn root development. One methodto identify and isolate the 3′ ends of cDNA of RNA particularlyexpressed in a specific tissue such as here the roots of plants, is theso-called READS analysis or Restriction-Enzyme digested cDNAs asdescribed e.g. by Prashar and Weismann or U.S. Pat. No. 5,712,126 (bothdocuments are herein incorporated by reference).

[0046] A cDNA reverse transcribed from RNA preferentially transcribed inroots of plants, such as corn plants, or 3′ ends of cDNAs identified byREADS differential display analysis as expressed preferentially in rootsof plants may be isolated and further characterized by e.g. nucleotidesequence determination; a full length cDNA may be isolated using e.g. 5′RACE (rapid amplification of cDNA ends) technology.

[0047] This cDNA or the 3′ end thereof may be used as a probe toidentify and isolate the region in the plant genome, comprising thenucleotide sequence encoding the corn root preferential mRNA.Alternatively, the genomic DNA can be isolated by e.g. inverse PCR usingoligonucleotides deduced from the cDNA sequence. Alternatively, TAIL-PCR(thermal assymetric interlaced PCR as described by Liu et al. (1995))using nested long specific oligonucleotides derived from the nucleotidesequence of the (5′ end) of the identified cDNA and a short arbitrarydegenerate primer may be used to isolate the genomic sequences flankingthe coding region.

[0048] Optionally, RNA probes corresponding to the cDNAs are constructedand used in conventional RNA-RNA in-situ hybridization analysis [seee.g., De Block et al. (1993), Anal. Biochem. 215: 86] of different planttissues, including the root tissue of interest, to confirm thepreferential presence of the mRNA produced by the endogenous plant genepresumed root preferential expression in those roots.

[0049] Once the corn root-prefential gene (i.e., the genomic DNAfragment, encoding the corn root-preferential mRNA from which thecorn-root preferential cDNA can be prepared) is obtained, the promoterregion containing the corn root-preferential promoter is determined asthe region upstream (i.e., located 5′ of) from the codon coding for thefirst amino acid of the protein encoded by the mRNA. It is preferredthat such promoter region is at least about 400 to 500 bp, at leastabout 1000 bp, about 1200 bp, about 1300 bp, or at least about 1500 to2000 bp, upstream of the start codon. For convenience, such promoterregion may not extend more than about 3000 to 5000 bp upstream of thestart codon. The size fragment may be partially determined by thepresence of convenient restriction sites. The actual cornroot-preferential promoter is the region of the genomic DNA upstream(i.e., 5′) of the region encoding the corn root-preferential mRNA. Achimeric gene comprising a corn root-preferential promoter operablylinked to the coding region of a marker gene will produce the markerprotein preferentially in the cells of the corn roots of transgenic cornplants, which can be assayed by conventional in situ histochemicaltechniques.

[0050] Examples of corn root-preferential genes from which cornroot-preferential promoters can be obtained, are genes, that encode amRNA which can be detected preferentially in corn roots and have a sizeof about 600 nts, from which a cDNA can be prepared that contains thecomplement of the nucleotide sequence corresponding to the nucleotidesequence of oligonucleotide GVK27 (SEQ ID No 7) and/or the complement ofthe nucleotide sequence of oligonucleotide GVK28 (SEQ ID No 8); and/orcontains the complement of the nucleotide sequence corresponding to theoligonucleotide GVK29 (SEQ ID No 9) and/or contains the complement ofthe nucleotide sequence corresponding to the oligonucleotide GVK30 (SEQID No 10) . Such corn root-prefential cDNA may contain each of theaforementioned sequences corresponding to oligonucleotides GVK27 andGVK28 as well as GVK29 or GVK30

[0051] Such a gene is the gene that encodes a corn root-preferentialtranscript from which a cDNA can be prepared that contains a nucleotidesequence encoding the protein with the amino acid sequence of SEQ ID 4and which may e.g. have the nucleotide sequence of SEQ ID No 3. Othercorn root-preferential genes are the genes that encode a cornroot-preferential mRNA from which a cDNA can be prepared that containsthe sequence of SEQ ID No 5 or SEQ ID 11, or that contains a nucleotidesequence encoding the protein comprising the amino acid sequence of SEQID 6.

[0052] One embodiment of a promoter of the present invention is apromoter contained in the 5′ regulatory region of a genomic clonecomprising a nucleotide sequence corresponding to the cDNA with thenucleotide sequence of any one of SEQ ID No 5, 6 or 11, e.g. the 5′regulatory region with the nucleotide sequence of SEQ ID No 2 from thenucleotide at position 11 to the nucleotide at position 1196 or a DNAfragment comprising the sequence of SEQ ID No 2 starting anywherebetween the nucleotide at position 11 to the nucleotide at postion 859,and ending at nucleotide position 1233 (just before the ATG translationstart codon) or a DNA fragment comprising the sequence of SEQ ID No 14from the nucleotide at position 1 to the nucleotide at position 1280.Such a promoter region comprises a corn root-preferential promoter ofthe invention and the 5′ untranslated leader region, and may be used forthe construction of root-preferential chimeric genes, particularly cornroot preferential chimeric genes. However, smaller DNA fragments can beused as promoter regions in this invention and it is believed that anyfragment of the DNA of SEQ ID No 2 which comprises at least the about400 basepairs upstream from the translation inititation codon can beused.

[0053] Artificial promoters can be constructed which contain thoseinternal portions of the promoter of the 5′ regulatory region of SEQ IDNo 1 or SEQ ID No 2 or SEQ ID 14 that determine the corn root-preferenceof this promoter. These artifical promoters might contain a “corepromoter” or “TATA box region” of another promoter capable of expressionin plants, such as a CaMV 35S “TATA box region” as described in WO93/19188. The suitability of promoter regions containing such artificialpromoters may be identified by their appropriate fusion to a reportergene and the detection of the expression of the reporter gene in theappropriate tissue(s) and at the appropriate developmental stage. It isbelieved that such smaller promoters and/or artificial promoterscomprising those internal portions of the 5′ regulatory region of SEQ IDNo. 1 or 2 that determine the corn root preference can provide betterselectivity of transcription in corn root cells and/or provide enhancedlevels of transcription of the transcribed regions of the cornroot-preerential chimeric genes of the invention. Such smaller portionsof the corn root preferential promoter regions of the invention mayinclude the nucleotide sequences which share a high homology between theGL4 and GL5 promoter regions such as: the nucleotide sequence of SEQ IDNo 2 from the nucleotide at position 1024 to the nucleotide at position1105 (having a 80% match with the nucleotide sequence of SEQ ID No 14from the nucleotide at position 435 to the nucleotide at position 510);the nucleotide sequence of SEQ ID No 2 from the nucleotide at position866 to the nucleotide at position 994 (having a 77% match with thenucleotide sequence of SEQ ID No 14 from the nucleotide at position 236to the nucleotide at position 358); the nucleotide sequence of SEQ ID No2 from the nucleotide at position 544 to the nucleotide at position 568(having a 96% match with the nucleotide sequence of SEQ ID No 15 fromthe nucleotide at position 198 to the nucleotide at position 222); thenucleotide sequence of SEQ ID No 2 from the nucleotide at position 1122to the nucleotide at position 1143 (having a 73% match with thenucleotide sequence of SEQ ID No 15 from the nucleotide at position 485to the nucleotide at position 510).

[0054] Besides the actual promoter, the 5′ regulatory region of the cornroot-preferential genes of this invention also comprises a DNA fragmentencoding a 5′ untranslated leader (5′UTL) sequence of an RNA locatedbetween the transcription start site and the translation start site. Itis believed that the 5′ transcription start site of the GL4 promoter islocated around position 1197 in SEQ ID No 2, resulting in a 5′UTL ofabout 30 nucleotides in length. It is believed that the 5′ transcriptionstart site of the GL5 promoter is located around position 1280 in SEQ IDNo 14, resulting in a 5′UTL of about 30 nucleotides in length It is alsobelieved that this region can be replaced by another 5′UTL, such as the5′UTL of another plant-expressible gene, without substantially affectingthe specificity of the promoter.

[0055] Thus, in another embodiment the invention provides a corn rootpreferential promoter or corn root preferential promoter regioncomprising a nucleotide sequence selected from the following group ofnucleotide sequences:

[0056] a nucleotide sequence comprising the nucleotide sequence of SEQID No 1 from the nucleotide at position 1 to the nucleotide at position338 or the nucleotide sequence of SEQ ID No 2 from the nucleotidesequence at position 11 to the nucleotide at position 1196;

[0057] a nucleotide sequence comprising the nucleotide sequence of SEQID No 14 from the nucleotide at position 1 to the nucleotide at position1280;

[0058] the nucleotide sequence of about an 400 bp to an about 1300 bpDNA fragment from (the 5′ end of) a corn root preferential gene encodinga mRNA, the mRNA having a size of about 600 nt, from which a cDNA can beprepared that contains the complement of the nucleotide sequencecorresponding to the nucleotide sequence of SEQ ID No 7, SEQ ID No 8,SEQ ID No 9, or SEQ ID No 10;

[0059] the nucleotide sequence of an about 400 bp to an about 1300 bpDNA fragment from the 5′ end of a corn root prefential gene encoding amRNA, the mRNA having a size of about 600 nt, from which a cDNA can beprepared that contains a nucleotide sequence encoding a polypeptide withthe amino acid of SEQ ID No 4 or 6;

[0060] the nucleotide sequence of an about 400 bp to an about 1300 bpDNA fragment from the 5′ end of a corn root preferential gene encoding amRNA, from which a cDNA can be prepared that comprises a nucleotidesequence having at least 75% or at least 80% or at least 90%, or atleast 95% sequence identity with the nucleotide sequence of any of SEQID No 3, 5 or 11 or is identical thereto;

[0061] the nucleotide sequence having at least 70% or 80% or 90% or 95%sequence identity to any of the nucleotide sequence mentioned under a),b), c),d), e), or f), particularly the nucleotide sequence mentionedunder a) or is identical thereto; or

[0062] the nucleotide sequence of an about 400 bp to an about 1300 bpDNA fragment hybridizing under stringent conditions with a DNA fragmenthaving the nucleotide sequence mentioned under a), b), c), d), e) or f),particularly the nucleotide sequence mentioned under a).

[0063] For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other, is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). The computer-assisted sequence alignment above can beconveniently performed using standard software program such as GAP whichis part of the Wisconsin Package Version 10.1 (Genetics Computer Group,Madison, Wis., USA) using the default scoring matrix with a gap creationpenalty of 50 and a gap extension penalty of 3.

[0064] Promoters and promoter regions of the invention may also compriseadditional elements known to improve transcription efficiency such asenhancers, introns, etc.

[0065] The invention further includes DNA molecules comprising the cornroot preferential promoters of the invention operably linked to one ormore heterologous regions coding for a biologically active RNA, peptideor protein. The promoters of the invention may be used to express anyheterologous coding region desired.

[0066] Thus in another embodiment of the invention, a chimeric gene isprovided comprising

[0067] a. a corn root preferential promoter region; comprising thenucleotide sequence selected from the group consisting of

[0068] i. the nucleotide sequence of SEQ ID No 1 from the nucleotide atposition 1 to the nucleotide at position 338 or the nucleotide sequenceof SEQ ID No 2 from the nucleotide at position 11 to the nucleotide atposition 1196;

[0069] ii. a nucleotide sequence comprising the nucleotide sequence ofSEQ ID No 14 from the nucleotide at position 1 to the nucleotide atposition 1280;

[0070] iii. the nucleotide sequence of about an 400 bp to an about 1300bp DNA fragment from (the 5′ end of) a corn root preferential geneencoding a mRNA, the mRNA having a size of about 600 nt, from which acDNA can be prepared that contains the complement of the nucleotidesequence corresponding to the nucleotide sequence of SEQ ID No 7 SEQ IDNo 8 or SEQ ID No 9 or SEQ ID No 10;

[0071] iv. the nucleotide sequence of an about 400 bp to an about 1300bp DNA fragment from the 5′ end of a corn root preferential geneencoding a mRNA, the mRNA having a size of about 600 nt, from which acDNA can be prepared that contains a nucleotide sequence encoding apolypeptide with the amino acid of SEQ ID No 4 or 6;

[0072] v. the nucleotide sequence of an about 400 bp to an about 1300 bpDNA fragment from the 5′ end of a corn root preferential gene encoding amRNA, from which a cDNA can be prepared that comprises a nucleotidesequence having at least 75% or 80% or 90% or 95% sequence identity withthe nucleotide sequence of any of SEQ ID No 3, 5 or 11 or is identicalthereto;

[0073] vi. the nucleotide sequence having at least 75% or 80% or 90% or95% sequence identity with the nucleotide sequence mentioned under i),ii), iii),iv), v), or vi), particularly the nucleotide sequencementioned under i) or is identical thereto; or

[0074] vii. the nucleotide sequence of an about 400 bp to an about 1300bp DNA fragment hybridizing under stringent conditions with a DNAfragment having the nucleotide sequence mentioned under i), ii), iii),iv), v) or vi), particularly the nucleotide sequence mentioned under a);

[0075] b. a DNA region of interest, which when transcribed yields abiologically active RNA; and

[0076] c. a DNA region comprising a 3′ transcription termination andpolyadenylation signal functional in plant cells.

[0077] The DNA region of interest, or the transcribed RNA may thusencode a protein or polypeptide, but may also encode biologically activeRNA, such as an antisense RNA, a sense RNA, or a dsRNA comprising bothsense and antisense RNA stretches capable of basepairing and forming adouble stranded RNA, as described in WO 99/53050 (incorporated herein byreference) usable for posttranscriptional gene silencing of a targetsequence.

[0078] To confer corn rootworm resistance, such as for exampleresistance to Diabrotica barberi, Diabrotica undecimpuncata, and/orDiabrotica virgifera, to plants, such as corn plants, in a rootselective or root preferential way, suitable candidate DNA regions to beoperably linked to the corn root selective promoters of the inventioninclude the mature VIP1Aa protein when combined with the mature VIP2Aaor VIP2Ab protein of PCT publication WO 96/10083; the corn rootwormtoxins of Photorhabdus or Xhenorhabdus spp., e.g., the insecticidalproteins of Photorhabdus luminescens W-14 (Guo et al., 1999, J. Biol.Chem. 274, 9836-9842); the CryET70 protein of WO 00/26378; theinsecticidal proteins produced by Bt strains PS80JJ1, PS149B1 andPS167H2 as described in WO 97/40162, particularly the about 14 kD andabout 44 kD proteins of Bt strain PS149B1; the Cry3Bb protein of U.S.Pat. No. 6,023,013; protease inhibitors such as the N2 and R1 cysteineproteinase inhibitors of soybean (Zhao et al., 1996, Plant Physiol. 111,1299-1306) or oryzastatine such as rice cystatin (Genbank entry S49967),corn cystatin (Genbank entries D38130, D10622, D63342) such as the corncystatin expressed in plants as described by Irie et al. (1996, PlantMol. Biol. 30, 149-157). Also included are all equivalents and variants,such as truncated proteins retaining insecticidal activity, of any ofthe above proteins.

[0079] In one embodiment of the invention, chimeric genes for conferringrootworm resistance in a root preferential way comprise a nucleotidesequence encoding an insecticidal secreted protein (ISP) fromBrevibacillus laterosporus, which is insecticidal when ingested by aninsect in combination with an ISP complimentary protein, such as anotherISP protein, as described in PCT application PCT/EP01/05702 published asWO 01/87931 (incorporated herein by reference; particularly DNAsequences SEQ ID No7 and 9 of WO 01/87931). The nucleotide sequencesencoding ISP protein may be a modified DNA.

[0080] The invention further provides methods for expressing a foreignDNA of interest preferentially in the roots of a plant, such as a cornplant, comprising the following steps: providing plant cells with thechimeric genes of the invention, which may be stably integrated in theirgenome, e.g. their nuclear genome, to generate transgenic cells; andregenerating plants from said transgenic cells.

[0081] A convenient way to provide plant cells with the chimeric genesof the invention is to introduce the DNA via conventional transformationmethods. It will be clear that actual method of transforming the plants,particularly cereal plants has little importance for the currentlydescribed methods. Several methods for introduction of foreign DNA intothe genome of plant cells are available in the art. These methodsinclude, but are not limited to, direct protoplast transformation (seee.g. for corn U.S. Pat. No. 5,792,936, incorporated herein byreference); Agrobacterium-mediated transformation (see e.g. for cornU.S. Pat. No. 6,074,877 or U.S. Pat. No. 6,140,553 incorporated hereinby reference); microprojectile bombardment, electroporation of compactembryogenic calli (see e.g. for corn U.S. Pat. No. 5,641,664incorporated herein by reference); or silicon whisker mediated DNAintroduction.

[0082] Operably linking the foreign DNA of interest to a corn rootpreferential promoter according to the invention may also be achieved byreplacing the DNA naturally associated with the corn root preferentialpromoter by homologous recombination with the DNA of interest, providedthat said DNA of interest comprises a homology region with the DNAnormally associated with the corn root preferential promoter. Methodsfor introducing DNA of interest into plant cell genome by homologousrecombination are available (e.g. U.S. Pat. No. 5,744,336 incorporatedherein by reference).

[0083] The obtained transformed plant can be used in a conventionalbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the chimeric gene for corn rootpreferential expression according to the invention in other varieties ofthe same or related plant species, or in hybrid plants. Seeds obtainedfrom the transformed plants contain the chimeric genes of the inventionas a stable genomic insert and are also encompassed by the invention.

[0084] It will be appreciated that the means and methods of theinvention are particularly useful for corn, but may also be used inother plants with similar effects, particularly in cereal plantsincluding corn, wheat, oat, barley, rye, rice, turfgrass, sorghum,millet or sugarcane plants.

[0085] The following non-limiting Examples describe the isolation ofcorn root preferential promoters, and the construction of chimeric genesfor selective expression in corn root plants. Unless stated otherwise inthe Examples, all recombinant DNA techniques are carried out accordingto standard protocols as described in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994)Current Protocols in Molecular Biology, Current Protocols, USA. Standardmaterials and methods for plant molecular work are described inPlantMolecular Biology Labfax (1993) by R. D. D. Croy, jointly published byBIOS Scientific Publications Ltd (UK) and Blackwell ScientificPublications, UK.

[0086] Throughout the description and Examples, reference is made to thefollowing sequences represented in the sequence listing.

[0087] SEQ ID No 1: nucleotide sequence of the about 400 bp corn rootpreferential promoter (GL4 promoter).

[0088] SEQ ID No 2: nucleotide sequence of the about 1200 bp corn rootpreferential promoter (GL4 promoter).

[0089] SEQ ID No 3: nucleotide sequence of the cDNA of the naturallyassociated mRNA transcribed under control of the corn root preferentialpromoter having the nucleotide sequence of SEQ ID No 1 (GL4).

[0090] SEQ ID No 4: amino acid sequence of the protein encoded by thecDNA GL4.

[0091] SEQ ID No 5: nucleotide sequence of the corn root preferentialcDNA GL5.

[0092] SEQ ID No 6: amino acid sequence of the protein encoded by thecDNA GL5.

[0093] SEQ ID No 7: oligonucleotide primer GVK 27.

[0094] SEQ ID No 8: oligonucleotide primer GVK28.

[0095] SEQ ID No 9: oligonucleotide primer GVK 29.

[0096] SEQ ID No 10: oligonucleotide primer GVK 30.

[0097] SEQ ID No 11: nucleotide sequence of corn root preferential cDNAGL12.

[0098] SEQ ID No 12: nucleotide sequence of plasmid pTWV011.

[0099] SEQ ID No 13: nucleotide sequence of plasmid pTWV018.

[0100] SEQ ID No 14: nucleotide sequence of the about 1300 bp corn rootpreferential promoter (GL5 promoter).

[0101] SEQ ID No 15: oligonucleotide primer GVK22.

[0102] SEQ ID No 16: oligonucleotide primer GVK23.

[0103] SEQ ID No 17: oligonucleotide primer GVK24.

[0104] SEQ ID No 18: oligonucleotide primer GVK25.

[0105] SEQ ID No 19: oligonucleotide primer GVK26.

[0106] SEQ ID No 20: oligonucleotide primer GVK31.

[0107] SEQ ID No 21: oligonucleotide primer GVK32.

[0108] SEQ ID No 22: oligonucleotide primer GVK33.

[0109] SEQ ID No 23: oligonucleotide primer GVK38.

[0110] SEQ ID No 24: oligonucleotide primer GVK39.

[0111] SEQ ID No 25: oligonucleotide primer GVK45.

[0112] SEQ ID No 26: oligonucleotide primer MDB285.

[0113] SEQ ID No 27: oligonucleotide primer MDB286.

[0114] SEQ ID No 28: oligonucleotide primer MDB363.

[0115] SEQ ID No 29: oligonucleotide primer MDB364.

[0116] SEQ ID No 30: oligonucleotide primer MDB552.

[0117] SEQ ID No 31: oligonucleotide primer MDB556.

EXAMPLES Example 1

[0118] Isolation of Root Preferential Corn cDNAs

[0119] RNA transcript tags which are expressed preferentially in maizeroots were identified by a differential RNA cDNA display known as READS(Prashar and Weismann, 1999, Methods in Enzymology 303:258- 272).

[0120] To this end, total RNA samples were prepared from differenttissues (roots, stems, leaves, . . . ) of corn plants, harvested atdifferent developmental stages (from 64 day old plants to adult plants).3′ end fragments of cDNA molecules digested by different endorestrictionnucleases were amplified using stringent PCR conditions andoligonucleotides as described by Prashar and Weismann (1999, supra) foreach of the samples.

[0121] Comparison of gel patterns of the 3′ end cDNA restrictionfragments generated for each of the RNA samples allowed a preliminaryidentification of fragments which appeared only in the corn root tissueRNA sample or were more prominent in root tissue RNA than in other corntissue. These 3′ end fragments were isolated and sequenced. Theirnucleotide sequence was compared against public and proprietarydatabases and only novel sequences were used in a further Northernanalysis using the RNA samples from different corn tissues as a driverand each of the isolated 3′ end cDNA fragments as a probe. Thehybridizing RNA transcripts were analyzed for size, abundance, andspecificity. The results for the 3′ end with the best specificity forexpression in corn roots are summarized in Table 1, arranged indescending order of specificity and abundance.

[0122] The 3′ ends which hybridized to the most abundant RNA transcriptswith the highest specific expression in corn roots (GL4; GL5 and GL12)were further analyzed.

[0123] For GL4 and GL5 full length cDNAs were isolated using the SMART™RACE cDNA amplification kit from CLONTECH Laboratories with nestedoligonucleotide primers GVK22 (SEQ ID No 15)/GVK23 (SEQ ID No 16) forGL4 and GVK24 (SEQ ID No 17)/GVK25 (SEQ ID No 18) for GL5 The nucleotidesequence of the full length cDNAs is represented in SEQ ID 3 and 5respectively. Comparison of both nucleotide sequences revealed that GL4and GL5 have about 89% sequence identity. TABLE 1 Estimated Specificityof Quantification of length of presence of the Identification of 3′hybridizing hybridizing hybridizing end used as probe transcript¹transcript² transcript GL4 18 About 600 Root selective GL5 15 About 600Root selective GL12 13 About 600 Root selective GL11 3 About 820 Rootselective GL3 1 About 1200 Root selective GL9 7 About 900 Rootpreferential GL7 2 About 700 Root preferential GL16 <2 About 1500 Lowexpression GL17 <2 About 650 Low expression GL6 — — No visiblehybridization

[0124] In both sequences, a small ORF could be identified (starting fromthe nucleotide of SEQ ID 3 at position 32 to the nucleotide at position319 for GL4; the nucleotide of SEQ ID 5 at position 27 to the nucleotideat position 307 for GL5). The amino acid sequences of the polypeptidesencoded by the ORFs are represented in SEQ ID No 4 or 6. The nucleotidesequence in the region encoding the ORF is more conserved between GL4and GL5 cDNA than elsewhere in the fragments.

[0125] Using the GL4 cDNA nucleotide sequence as a query, a nucleotidesequence has been identified with 91% sequence identity in the322-nucleotide overlap (SEQ ID No 19 from WO 00/32799). It has not beendescribed that SEQ ID No 19 from Zea mays as described in WO 00/32799 istranscribed in a root selective or root preferential way. Further, anucleotide sequence (clone MEST23-CO7.T3 from a seedling and silk cDNAlibrary) has been identified having 99% sequence identity over 582 nt.

[0126] Using the GL5 cDNA nucleotide sequence as a query, a nucleotidesequence has been identified with 85% sequence identity with FtsZ1related sequence from Zea mays (A47325 in Geneseq). It has not beendescribed that this sequence is transcribed in a root selective or rootpreferential way. Further, a nucleotide sequence (clone MEST523-G12 (3′)from a seedling and silk cDNA library) has been identified having 100%sequence identity over 525 nt.

Example 2

[0127] Isolation of Corn Root Preferential Promoter Regions of the GeneTranscribing a mRNA, the cDNA of which Corresponds to GL4 or GL5 cDNA.

[0128] The genomic fragments upstream of the nucleotide sequencescorresponding to GL4 cDNA and GL5 cDNA sequences, comprising thepromoter region were isolated using Thermal Asymmetric Interlaced PCR asdescribed by Liu et al (1995, The Plant Journal 8(3): 457-463)

[0129] Corn genomic DNA for use as the initial template DNA was purifiedas described by Dellaporte et al. The sequence of the specific nestedoligonucleotides used for TAIL-PCR to isolate the genomic fragmentslocated upstream of the genomic DNA sequences corresponding to GL4 cDNAsequences are represented in SEQ ID No 7 (GVK27), SEQ ID No 8 (GVK28)and SEQ ID No 9 (GVK 29); the aspecific degenerate primers used wereeach of the 6 degenerate primers MDB285, MDB286, MDB363, MDB364, MDB552or MDB556 in separate reactions. PCR conditions were as described in Liuet al (1995, supra).

[0130] A genomic fragment of about 400 bp (corresponding to theamplification product obtained with the primer pair (GVK29/MDB285) wasisolated, cloned in pGEM-T Easy ® and sequenced.

[0131] Based on the nucleotide sequence of the about 400 bp fragment,new specific nested primer oligonucleotides (GVK 31/SEQ ID No 20;GVK32/SEQ ID No 21 and GVK33/SEQ ID No 22) were designed and used inconjunction with the above mentioned degenerated primers to isolate theadjacent DNA regions further upstream of the isolated promoter regionfragment. This resulted in isolation of an about 350 nt DNA fragment(corresponding to the amplification product obtained with the primerpair GVK33/MDB286) In a third round, the adjacent upstream DNA fragmentof about 800 nt was isolated using new set of specific nestedoligonucleotides GVK33/SEQ ID No 22; GVK38/SEQ ID No 23 and GVK39/SEQ IDNo 24 in conjunction with the above mentioned degenerated primers(corresponding to the amplification product using GVK39 and MDB363).

[0132] To confirm the continuity of the isolated genomic upstreamfragments, the complete 1200 bp DNA fragment was amplified using GVK29and GVK45 (SEQ ID 25) primers and cloned in pGEM-T Easy®. The completenucleotide sequence of the about 1200 bp upstream DNA fragment isrepresented in SEQ ID 2.

[0133] Primers GVK 27, GVK28 and GVK30 (SEQ ID No 10) were used inconjunction with the above mentioned degenerated primers. An about 1300nt fragment was amplified having the sequence of SEQ ID 14(corresponding to the amplification product by MDB364 and GVK30).

Example 3

[0134] Construction of Chimeric Genes using the Isolated GL4/GL5 CornRoot Preferential Promoter Regions.

[0135] The following chimeric ISP1A/ISP2A constructs under the controlof GL4 promoter region or under control of the GL5 promoter region weremade using standard recombinant DNA methods:

[0136] GL4::ISP1A comprising the following DNA fragments:

[0137] the GL4 promoter region (SEQ ID No 2);

[0138] a DNA fragment encoding the isp1A protein of Brevibacilluslaterosporus (complement of the nucleotide sequence of SEQ ID 12 fromthe nucleotide at position 2003 to the nucleotide at position 4511);

[0139] the 3′ end fragment of the 35S transcript (complement of thenucleotide sequence of SEQ ID No 12 from the nucleotide at position 1767to the nucleotide at position 1991).

[0140] GL4::ISP2A comprising the following DNA fragments:

[0141] the GL4 promoter region (SEQ ID No 2);

[0142] a DNA fragment encoding the isp2A protein of Brevibacilluslaterosporus (complement of the nucleotide sequence of SEQ ID 12 fromthe nucleotide at position 6001 to the nucleotide at position 7228); and

[0143] the 3′ end fragment of the 35S transcript (complement of thenucleotide sequence of SEQ ID No 12 from the nucleotide at position 5765to the nucleotide at position 5989).

[0144] GL5::ISP1A comprising the following DNA fragments:

[0145] the GL5 promoter region (complement of the nucleotide sequence ofSEQ ID No 13 from the nucleotide at position 4518 to the nucleotide atposition 5822);

[0146] a DNA fragment encoding the ispA1 protein of Brevibacilluslaterosporus (complement of the nucleotide sequence of SEQ ID 13 fromthe nucleotide at position 2003 to the nucleotide at position 4511); and

[0147] the 3′ end fragment of the 35S transcript (complement of thenucleotide sequence of SEQ ID No 13 from the nucleotide at position 1767to the nucleotide at position 1991).

[0148] GL5::ISP2A comprising the following DNA fragments:

[0149] the GL5 promoter region (complement of the nucleotide sequence ofSEQ ID No 13 from the nucleotide at position 8628 to the nucleotide atposition 7324);

[0150] a DNA fragment encoding the isp2A protein of Brevibacilluslaterosporus(complement of the nucleotide sequence of SEQ ID 12 from thenucleotide at position 6090 to the nucleotide at position 7317); and

[0151] the 3′ end fragment of the 35S transcript (complement of thenucleotide sequence of SEQ ID No 12 from the nucleotide at position 5765to the nucleotide at position 5989).

Example 4

[0152] Expression Analysis of Chimeric Genes Comprising the GL4 and GL5Promoters in Stably Transformed Corn Plants.

[0153] The chimeric genes described in Example 3 were introduced in aT-DNA vector along with a chimeric bar gene (such as described in U.S.Pat. No. 5,561,236) to yield pTWV011 (GL4 promoter constructs) andpTWV018 (GL5 promoter constructs). These T-DNA vectors were introducedin Agrobacterium tumefaciens containing the helper Ti-plasmid pGV4000 orpEHA101.

[0154] Corn plants stably transformed with the mentioned chimeric geneswere obtained using the Agrobacterium-mediated transformation techniquedescribed in U.S. Pat. No. 6,140,553.

[0155] Corn plants stably transformed with the mentioned chimeric genesmay also be obtained using direct DNA delivery to corn protoplast asdescribed in U.S. Pat. No. 5,767,367.

[0156] RNA was isolated from root, leaf and stem tissue of these cornplants (grown either in vitro or in vivo) and submitted to Northernanalysis using a ISP1A specific probe. The individual results arerepresented in Table 3.

[0157] In summary, after correction for loading by hybridization withribosomal RNA probe, the GL5 promoter region on average initiated 11times more transcription in roots than in leaves and 19 times more inroots than in stems.

[0158] The GL4 promoter region on average initiates 2 times moretranscription in roots than in leaves and more than 10 times more inroots than in stems (although individual transformants may exhibit amore pronounced corn root selective expression pattern). TABLE 2 Summaryof GL4/GL5 transcription data mean n = 3 SD Gl4 promoter root 1.25 1.18root/leaf 2 leaf 0.61 0.73 root/stem >10 stem <0.06 Gl5 promoter root1.32 0.24 root/leaf 11 leaf 0.12 0.05 root/stem 19 stem 0.07 0.03

[0159] TABLE 3 Summary of Northern analysis data Isp1a mRNA (pg/μg Cornline tot. RNA) pTWV018 GL5 promoter 1 G2ZM3598- root in vitro 1.07 004 2root 1.57 3 leaf {close oversize brace} in vivo 0.15 root/leaf 10.2 4stem 0.06 root/stem 26.2 5 G2ZM3595- root in vitro 2.17 014 6 root 1.107 leaf {close oversize brace} in vivo 0.15 root/leaf 7.3 8 stem 0.10root/stem 11 9 G2ZM3595- root in vitro 1.39 018 10 root 1.28 11 leaf{close oversize brace} in vivo 0.07 root/leaf 18.3 12 stem 0.04root/stem 32 13 G2ZM3596- root in vitro 0.73 016 14 root <0.06 15 leaf{close oversize brace} in vivo <0.06 root/leaf nd 16 stem <0.06root/stem nd pTWV011 GL4 promoter 1 G2ZM3592- root in vitro 0.53 029 2root 0.72 3 leaf {close oversize brace} in vivo 0.08 root/leaf 9 4 stem<0.06 root/stem >12 5 G2ZM3592- root in vitro 1.45 030 6 root 2.60 7leaf {close oversize brace} in vivo 1.45 root/leaf 1.8 8 stem <0.06root/stem >43 9 G2ZM3593- root in vitro 0.34 002 10 root 0.43 11 leaf{close oversize brace} in vivo 0.31 root/leaf 1.4 12 stem <0.06root/stem >7.2

Example 5

[0160] Expression Analysis of Chimeric Genes Comprising the GL4 and GL5Promoters in Progeny of Stably Transformed Corn Plants.

[0161] Nine transgenic T0 lines of each of the GL4 and GL5 promotercontaining corn plants of Example 4 were crossed with untransformed B73,and the T1 plants were analyzed by Southern blot, Northern blot and byELISA assay for the presence of ISP1 mRNA or protein in various plantparts.

[0162] Southern analysis was performed at the V4 stage to determine thecopy number of the transgenes. All analyzed events were single copyevents except two lines that contained 2 copies of the transgene.

[0163] Northern analysis was performed on RNA derived from root, leafand stem material obtained at the V11-V 13 stage as in Example 4.Transcript levels were quantified using image quant analysis. Acorrection for loading differences was performed using a ribosomalprobe.

[0164] For the plants with the GL4 promoter containing transgene, isp1mRNA was estimated between 0.17 to 0.74 pg/μg total RNA. The averageisp1a mRNA level in roots (n=9) was 0.42 pg/μg total RNA (SE=0.18). Theaverage ratio (n=9) of isp1a mRNA in root versus leaf is >6.3. Theaverage ratio (n=8) of isp1a mRNA in root versus stem is >7.1. Noexpression was seen in stem, except for one sample where the ratioroot/stem was 1.9.

[0165] For the plants with the GL5 promoter containing transgene, isp1mRNA was estimated between 0.95 to 2.55 pg/μg total RNA. The averageisp1a mRNA level in roots (n=9) was 1.57 pg/μg total RNA (SE=0.53). Theaverage ratio (n=9) of isp1a mRNA in root versus leaf is >17.6. Theaverage ratio (n=8) of isp1a mRNA in root versus stem is >26.6. Noexpression was observed in stem.

[0166] Root, leaf and stem material, harvested at the V8 stage, wasanalyzed at the protein level for the presence of ISP1a protein byELISA. Two plants per event were analyzed. As a negative control, rootleaf and stem for wtB73 was checked for ISP1A protein. No ISP1A proteinwas detected in the control experiments.

[0167] For all events no ISP1A protein expression was detected in leavesor stem. The mean value of levels of ISP1A protein detected in rootswere:

[0168] 0.07 μg/ml corresponding to about 0.024% of total protein level(n=18) for roots of plants containing the GL4 promoter driven transgene.

[0169] 0.12 μg/ml corresponding to about 0.041% of total protein level(n=18) for roots of plants containing the GL5 promoter driven transgene.

[0170] Root, leaf, stem and pollen material, harvested at the floweringstage, was analyzed at the protein level for the presence of ISP1aprotein by ELISA. One plant per event was analyzed. As a negativecontrol, root, leaf, stem and pollen material for wtB73 was checked forISP1A protein. No ISP1A protein was detected in the control experiments.

[0171] For all events, no ISP1A protein expression was detected inpollen.

[0172] Mean ISP1A Protein level detected in root, leaf and stem ofplants containing the GL4 promoter driven transgene:

[0173] mean value (n=9)

[0174] root: 0.286 μg isp1a/ml ˜0.116% isp1a of tot. protein level

[0175] leaf: 0.018 μg isp1a/ml ˜0.0022% isp1a of tot. protein level (6%of root level)

[0176] stem: 0.020 μg isp1a/ml ˜0.0043% isp1a of tot. protein level (7%of root level)

[0177] Mean ISP1A Protein level detected in root, leaf and stem ofplants containing the GL4 promoter driven transgene

[0178] mean value (n=9):

[0179] root: 0.265 μg isp1A/ml ˜0.142% isp1a of tot. protein level

[0180] leaf: 0.013 μg isp1A/ml ˜0.0015% isp1a of tot. protein level (5%of root level)

[0181] stem: 0.024 μg isp1A/ml ˜0.0058% isp1a of tot. protein level (9%of root level)

[0182] At seed setting, ISP1A protein level was determined in kernels ofthe transgenic plants. No ISP1A protein could be detected in seed of thetransgenic plants, nor in the wt B73 control.

1 32 1 378 DNA Zea mays misc_feature (1)..(338) promoter 1 tactacagataacacgacag ttaacgagcg ggtatgggtt gttttccttg agcactgttg 60 ttctctagaatctctgaatc tctctctgtc ttgatgacac cgagcggaaa tagcagttgg 120 aagaggtgattgggcttcag cgcgcgatcc aacccaagtg ggttccacaa cgtgaacctc 180 atgcagcttaaaatacagcc agttgtgatc catctgccac agctgtttct acctcagatg 240 tgctacacagtgtattacct gtttctacct cgcagatgtg ctacacagtt gcttatgact 300 gcctataaaatggccgggat cggtgaggct gctggaacca aggagagaga gcatatatat 360 ccaccgatccatggcatg 378 2 1236 DNA Zea mays misc_feature (11)..(1196) promoter 2cgggatcccg gctttctgca ctggacgtag tgtactttat acttgaaact tgtataaatt 60tgtgtctttt atactccctc agtttgaaat atagttcttt ctagcctctt tttttccgtc 120cacactcatt tgaatgataa taaatataga tatacataca aactatattc ataggttaat 180taataaatgt atatttagtc taaaatgaaa tatattttac ccatcgtatt ccttatgcat 240gaaatgttga tctacttgtc tgatggaaaa atactatgac gttgttgtac cagaccgcac 300ctaaatcaaa ctgttttcag agatggccat tctattattg tagatttgtg atacgtacga 360tgtacttttt tatccataaa ataccgtacc attatgatat ggatatcttg atgagaggga 420ctcattatct ctctctatat atataaacac ctatatatca aacaggcatc aagaaaaata 480gatgattttt ttttctgaag tagagtgaca gaagcagctg aagtgtgagt ctttttgttt 540caattttata atgtgtaaag aaaatgacgc caatgaaata tgtgtctggg ctgacgtgtt 600gtttggtgaa agccaatttt gttgtatata ggggggccag agcccagttg tatttgttgc 660ccggactggc gccaaaaaaa aaaatccgga tagtactatt ccgctaactg tgtcacactt 720tatctaaaat tagtcatcca aattaaagaa ctaaccttag atacaaaaaa ttaaacaaag 780tatgacaagt taggtagcaa actaaactaa agaggataac acaacagtta accgtcgacg 840tgcgcggcct gaatttacta ctacagataa cacgacagtt aacgagcggg tatgggttgt 900tttccttgag cactgttgtt ctctagaatc tctgaatctc tctctgtctt gatgacaccg 960agcggaaata gcagttggaa gaggtgattg ggcttcagcg cgcgatccaa cccaagtggg 1020ttccacaacg tgaacctcat gcagcttaaa atacagccag ttgtgatcca tctgccacag 1080ctgtttctac ctcagatgtg ctacacagtg tattacctgt ttctacctcg cagatgtgct 1140acacagttgc ttatgactgc ctataaaatg gccgggatcg gtgaggctgc tggaaccaag 1200gagagagagc atatatatcc accgatccat ggcatg 1236 3 592 DNA Artificial cDNAof GL4 transcript 3 caaggagaga gagcatatat atccaccgat catgatgaagggtggcagca agaaggaagt 60 ggccggtgcg gcggcggtgg tggccatact gctggttctgcagctgatgg cagctccacc 120 gacggccatg gccgcccgct cgccgcgcgg agccgtgccggatggctccc tcgccacgac 180 gcccaaggtg acgatgctgt cagccacgct gtgctacacgggggagacat gcaaatacat 240 tacctgcctc actcctgctt gctcctgtaa ctatgatgatcgtcgctgct acatcatatt 300 tactcctgct gctgcttgag gccattctgt gtacgtgaatgaagccacta ctactctcac 360 acagcatgcg ccggccgacg acgtgcgtac gtatatatatacgctctacc tcgtgagctt 420 ttgttcgagt gatacgtgtt tcaaggcatc catccatccatggatgctta tgtacgtata 480 tgtgttagtc gtgtgtcagg caaccgggca gcagaagggggtgttgtatt atatatattn 540 acgtcttctg gtgattaaat aataaagggg ggcatgttggatgtgtgcaa aa 592 4 95 PRT Zea mays 4 Met Met Lys Gly Gly Ser Lys LysGlu Val Ala Gly Ala Ala Ala Val 1 5 10 15 Val Ala Ile Leu Leu Val LeuGln Leu Met Ala Ala Pro Pro Thr Ala 20 25 30 Met Ala Ala Arg Ser Pro ArgGly Ala Val Pro Asp Gly Ser Leu Ala 35 40 45 Thr Thr Pro Lys Val Thr MetLeu Ser Ala Thr Leu Cys Tyr Thr Gly 50 55 60 Glu Thr Cys Lys Tyr Ile ThrCys Leu Thr Pro Ala Cys Ser Cys Asn 65 70 75 80 Tyr Asp Asp Arg Arg CysTyr Ile Ile Phe Thr Pro Ala Ala Ala 85 90 95 5 535 DNA Artificial cDNAof GL5 transcript 5 ccaatcagat agagagcata gtcgatcatg aagggtggcaagaagaaagt ggccggtgcg 60 gtggtggcca tactgctggt tntgcagctc atggcagctccaccgacggc catggccgcc 120 cgctcgccgc gcggagccgt gccggatggc tccctcgccacgacgcccaa ggtgacgatg 180 ctgtcggcca cgctgtgcta cacgggggag acatgcaaatacattggctg cctcactcct 240 gcttgctcct gcaactatag tgatcgtcta tgctacatcatatttactcc tgttgcttga 300 ggccattccg cgaagccaca actcttacaa tatgcatgcgccggccgacg acgacgcgcg 360 ctgcctctcg tgagcttctg ttcaagtgat gcatgtttcaaggcatccat ggatgcttta 420 cgtatatgcg tattaattag ccgtgtcagg gaaccggacagaagggggtg ttgttttata 480 tttacgtctt ctggtgatca aataaagggg aaatatatgttggatgtgtg caaaa 535 6 90 PRT Zea mays 6 Met Lys Gly Gly Lys Lys Glu ValAla Gly Ala Val Val Ala Ile Leu 1 5 10 15 Leu Val Leu Gln Leu Met AlaAla Pro Pro Thr Ala Met Ala Ala Arg 20 25 30 Ser Pro Arg Gly Ala Val ProAsp Gly Ser Leu Ala Thr Thr Pro Lys 35 40 45 Val Thr Met Leu Ser Ala ThrLeu Cys Tyr Thr Gly Glu Thr Cys Lys 50 55 60 Tyr Ile Gly Cys Leu Thr ProAla Cys Ser Cys Asn Tyr Ser Asp Arg 65 70 75 80 Leu Cys Tyr Ile Ile PheThr Pro Val Ala 85 90 7 24 DNA Artificial oligonucleotide primer GVK27 7gctgacagca tcgtcacctt gggc 24 8 25 DNA Artificial oligonucleotide primerGVK28 8 gctgcagaac cagcagtatg gccac 25 9 27 DNA Artificialoligonucleotide primer GVK29 9 catgccatgg atcggtggat atatatg 27 10 25DNA Artificial oligonucleotide primer GVK30 10 catgccatgg atcgactatgctctc 25 11 164 DNA Artificial cDNA of 3′ end of the GL12 transcript 11ttttacacac atccaacatg ccccccttta ttatttaatc accanaagac gtnaatntnt 60nttattcaac accccctttt gctgcccngg tgnctnacac accactaaca catttccgtt 120cataancatt catgctgggc cgcgctgtgc ccgacanctt aaac 164 12 8514 DNAArtificial plasmid pTWV011 12 cggcaggata tattcaattg taaatggctccatggcgatc gctctagagg atcttcccga 60 tctagtaaca tagatgacac cgcgcgcgataatttatcct agtttgcgcg ctatattttg 120 ttttctatcg cgtattaaat gtataattgcgggactctaa tcataaaaac ccatctcata 180 aataacgtca tgcattacat gttaattattacatgcttaa cgtaattcaa cagaaattat 240 atgataatca tcgcaagacc ggcaacaggattcaatctta agaaacttta ttgccaaatg 300 tttgaacgat ctgcttcgga tcctagacgcgtgagatcag atctcggtga cgggcaggac 360 cggacggggc ggtaccggca ggctgaagtccagctgccag aaacccacgt catgccagtt 420 cccgtgcttg aagccggccg cccgcagcatgccgcggggg gcatatccga gcgcctcgtg 480 catgcgcacg ctcgggtcgt tgggcagcccgatgacagcg accacgctct tgaagccctg 540 tgcctccagg gacttcagca ggtgggtgtagagcgtggag cccagtcccg tccgctggtg 600 gcggggggag acgtacacgg tcgactcggccgtccagtcg taggcgttgc gtgccttcca 660 ggggcccgcg taggcgatgc cggcgacctcgccgtccacc tcggcgacga gccagggata 720 gcgctcccgc agacggacga ggtcgtccgtccactcctgc ggttcctgcg gctcggtacg 780 gaagttgacc gtgcttgtct cgatgtagtggttgacgatg gtgcagaccg ccggcatgtc 840 cgcctcggtg gcacggcgga tgtcggccgggcgtcgttct gggtccatgg ttatagagag 900 agagatagat ttatagagag agactggtgatttcagcgtg tcctctccaa atgaaatgaa 960 cttccttata tagaggaagg gtcttgcgaaggatagtggg attgtgcgtc atcccttacg 1020 tcagtggaga tgtcacatca atccacttgctttgaagacg tggttggaac gtcttctttt 1080 tccacgatgc tcctcgtggg tgggggtccatctttgggac cactgtcggc agaggcatct 1140 tgaatgatag cctttccttt atcgcaatgatggcatttgt aggagccacc ttccttttct 1200 actgtccttt cgatgaagtg acagatagctgggcaatgga atccgaggag gtttcccgaa 1260 attatccttt gttgaaaagt ctcaatagccctttggtctt ctgagactgt atctttgaca 1320 tttttggagt agaccagagt gtcgtgctccaccatgttga cgaagatttt cttcttgtca 1380 ttgagtcgta aaagactctg tatgaactgttcgccagtct tcacggcgag ttctgttaga 1440 tcctcgattt gaatcttaga ctccatgcatggccttagat tcagtaggaa ctaccttttt 1500 agagactcca atctctatta cttgccttggtttatgaagc aagccttgaa tcgtccatac 1560 tggaatagta cttctgatct tgagaaatatgtctttctct gtgttcttga tgcaattagt 1620 cctgaatctt ttgactgcat ctttaaccttcttgggaagg tatttgatct cctggagatt 1680 gttactcggg tagatcgtct tgatgagacctgctgcgtag gaacgcggcc gcgtatacgt 1740 atcgatatct tcgaattcat atgcatgatctggattttag tactggattt tggttttagg 1800 aattagaaat tttattgata gaagtattttacaaatacaa atacatacta agggtttctt 1860 atatgctcaa cacatgagcg aaaccctataggaaccctaa ttcccttatc tgggaactac 1920 tcacacatta ttatggagaa aatagagagagatagatttg tagagagaga ctggtgattt 1980 cagcgtgtcc aagcttgcta gcctcagtccacagcgaaga tcctcaccac agcggtcctg 2040 gtggagcccc acaggtcgtc cttgcaggacagggtgtaca ggaaggacct gtcggacttc 2100 acgttcttga agtccaggtt gatcctgttagcgttcttgc cggaaccgga gtccagcacg 2160 gtcttggtct gctcaccgat caccttgtaggtgaaggaga aggtacctgg ggtctgggta 2220 atcagtgggg agtcgatgaa gtaggtagcgtcgtggtact tcacggccag tgggtcaccg 2280 aagaagtcga agtcaccctc gatgatgtcgaactgtggca ctgggtcctt gtactcgatc 2340 tcctcagcac ccacctccac gaaggacacgtcgtcccagt acacgttggt ctggccgtca 2400 cccttgatgg agatggtgtt gatctcgttgccctccaggt ttggcaccag gatgttgatc 2460 ctctggtaac ccacgtggtc cagggtgatgttgtcggtca cgatggactc ctgcttaccg 2520 tcgatctcga tgtccacgga caccttggagtcagccttca tgtacaggga cacgtagtag 2580 tcgatgttct tcttcagctt gttcttggactcggtggaca gctcggtgaa agcacccttg 2640 ttagcggacc tgtactgctt cttaccggtgttaccaccgt tcaccacgta ggtgtagtac 2700 cagttgccga tcttggtgtt gtcggactcaccaccgtcgt acagggtgga ggtctttatt 2760 gtgaagttca tcttcggttc gagcttcacgtcgtacaggt tcttcacgtc cttgaaggaa 2820 ccggtggagt cgttcagctg cttcctgatctgcttagcgg tgtactcgtc cacgtaggac 2880 tgcacggaag cctcgaagat cggctggtcgttgtagaaca gcaggccgtc cttctcctcg 2940 atctcgtctg ggtaggccag cttcagggcctccttcacgg ataaatttgg ggtcttgtcc 3000 tctgggttgg tgtagtcctt agcagccaccctcttctcgg acatctggtt gccgttgtcg 3060 acgatgatgc tggcggtctt ggcggagatctcgtcggtga tgccgttcca gtcaccagcg 3120 atggtgatgt tgccgttggt gtccttgatggcgtacttgc cctccacctg gtcggtttcg 3180 agcaggatcg gcttcttgtt ggacaggtaggtgttcagct gctccttgtt cagtgggatc 3240 ggcctggagt tgaagtcgtc catcgtgttgatggcgatgc cgttcttgcc cttctctggg 3300 taggactggt ctggcaggat ggtcagagcggtggtgttct ccttggcctt gatggtgccg 3360 atggtggtgc cgtccaggat gaaggaggtggttggcttgg tctcgtagat agcaccggtg 3420 cccacgttgt tgtacctcac gttggcgttcaggtaggcgg actcagcacc gttgatgtgg 3480 gtgccgtcgt tggtggcgga gccccactcgttggccacgg tctcggagtg ctggtagttc 3540 acggacacgc cgaaggatgg acccagaccggaccaaccag cgttcacgtt cacgccctcg 3600 gtgttggtgt aggaccagtt ggtggactgggaggactcca cggagtggga caggtcctcg 3660 ttcttggaca ggatcacctt ttcgagggacacgttcacgg atgggaaagc agccaccagt 3720 gggttgaagg tctccttggc gttggacagtggcatgtccc tagcagcctt ctcgtagtcg 3780 gagtatgggt cgcccacggt gtgggcttcgagtgggttgc tggtgaactt ctggtagccc 3840 ttggaggcca gggagtcgtc ccacttcacggccaccttgt tctggatggt gtagccgttc 3900 tcctcccaca cgtccgggat ggagtcgccgtcggtgtcgg tgtcctcgtc gatgtccctc 3960 ttggtcttct gggtgaacag gttggtcttggaggccttct tcaggaacac ctgggtctcc 4020 ttcttgttga actctgggtt cctcagctcgtcctgctgca cctgctggga gtggttctgg 4080 gagtcgatct tgaacagctt cagctccttgaagatcttgt tgtcgatgtg cagggcgtcg 4140 tcggactggt actcgatctt gatctgcaccagctggccct tttcgaggtg cacggactgc 4200 ttgttgttgc ccttctcgga gatcaccttgccgtccagct cgatgatggc gttctcgtcg 4260 tcggacagct tgaaggtgaa gtcaccagtggcggaggact ggatcaggcc gatccagcgg 4320 atggagtggt actcctggtg cttctggtccacgagggtgt tagcggtctg ctggtcgtag 4380 atcagggtgt tgtccctggt tggagcgaacagggtcaggt cgttgaagtc cttgcccttg 4440 aagtagtagc ccagcaggcc ctccctgtcgatctggttgt ccttcgaagc ctgggtggtg 4500 gtggcgatag ccatggatcg gtggatatatatgctctctc tccttggttc cagcagcctc 4560 accgatcccg gccattttat aggcagtcataagcaactgt gtagcacatc tgcgaggtag 4620 aaacaggtaa tacactgtgt agcacatctgaggtagaaac agctgtggca gatggatcac 4680 aactggctgt attttaagct gcatgaggttcacgttgtgg aacccacttg ggttggatcg 4740 cgcgctgaag cccaatcacc tcttccaactgctatttccg ctcggtgtca tcaagacaga 4800 gagagattca gagattctag agaacaacagtgctcaagga aaacaaccca tacccgctcg 4860 ttaactgtcg tgttatctgt agtagtaaattcaggccgcg cacgtcgacg gttaactgtt 4920 gtgttatcct ctttagttta gtttgctacctaacttgtca tactttgttt aattttttgt 4980 atctaaggtt agttctttaa tttggatgactaattttaga taaagtgtga cacagttagc 5040 ggaatagtac tatccggatt tttttttttggcgccagtcc gggcaacaaa tacaactggg 5100 ctctggcccc cctatataca acaaaattggctttcaccaa acaacacgtc agcccagaca 5160 catatttcat tggcgtcatt ttctttacacattataaaat tgaaacaaaa agactcacac 5220 ttcagctgct tctgtcactc tacttcagaaaaaaaaatca tctatttttc ttgatgcctg 5280 tttgatatat aggtgtttat atatatagagagagataatg agtccctctc atcaagatat 5340 ccatatcata atggtacggt attttatggataaaaaagta catcgtacgt atcacaaatc 5400 tacaataata gaatggccat ctctgaaaacagtttgattt aggtgcggtc tggtacaaca 5460 acgtcatagt atttttccat cagacaagtagatcaacatt tcatgcataa ggaatacgat 5520 gggtaaaata tatttcattt tagactaaatatacatttat taattaacct atgaatatag 5580 tttgtatgta tatctatatt tattatcattcaaatgagtg tggacggaaa aaaagaggct 5640 agaaagaact atatttcaaa ctgagggagtataaaagaca caaatttata caagtttcaa 5700 gtataaagta cactacgtcc agtgcagaaagccgggatcc ccgggctagg cgcgccatat 5760 gcatgatctg gattttagta ctggattttggttttaggaa ttagaaattt tattgataga 5820 agtattttac aaatacaaat acatactaagggtttcttat atgctcaaca catgagcgaa 5880 accctatagg aaccctaatt cccttatctgggaactactc acacattatt atggagaaaa 5940 tagagagaga tagatttgta gagagagactggtgatttca gcgtgtccaa gcttgctagc 6000 ctcacttggt cagcagggta gcgtccactacgtacctctt cacacccttg atcaccacct 6060 cggtgatctt gtcgatgtgg tagttggagtccttgtcgat caggatctcc ttctcggaag 6120 cgaagccacc gatagcggac aggtaggcaccagtggaacc ctttggcacc tgcagcctca 6180 ggatgaactt cctggagccg aaagcggacagcctctcgga ggacagggag gtggacatgt 6240 aacccttgtc ctccttcatg gtgttcaggaacttctcctc catctccttc agggatggca 6300 gtgggtcgga aatctggtaa ccgaactcggccataccgca ccacctgtac acggtgatgt 6360 tctctgggat cggctgcttc tccagagcctcggagatgtt cttgatctgg gtgtccagct 6420 tctcgttgcc ggagccaccc tggttccgcaggtagtcgtt gatctgcttg tagtcctgcc 6480 tagcgtagcc gtccagggcc tccctctgtgggtcggtcag gttcttagcc caaccctcgt 6540 agttcttcat gccccacctg tgagcctcggcgttgatgtc gttcttgaag tccagggact 6600 tcttcagggt accctggatc tgcaggcactcgtaaccctt cttcaccacc ttggagatgt 6660 tgtccacgtg cagcacgtag ccgttgtcgatcagcatctt gtactcgttg ttgttcagga 6720 tcacaccagc cttggtcggg atggtggagcccttgccgga tggcacggtc acttggagga 6780 tgatcctctc cttggaggac acgttctgagcagtcaggtg ggtgtccagg taggagtcga 6840 acttgatgtc cttgcccagg aactgctccttgaactgggc ctgcacgtcg gtgttgatgg 6900 tgttgccctc ggtcagtggc ttgttaaatccgatggtgga tggctccacg ttcttgtagg 6960 tcacgatgga ggaggacagg ttggccttgtcgaacatctt gtcgatctcc ttcaggtcct 7020 tgatctcgtc ctcgaaggag ccagccatgctgaaggtgat ctccttgtag ttcttcttga 7080 tgtcgttctt gttgtccagg aaattattcatacgcgtctt ctcggtcacg gtcagcttcc 7140 actccttctc cttctccttg ccccactccttggccttctc cttgtcctcc ttgaagtcct 7200 cggtgttgtt ggtggtcttc actagtgccatggatcggtg gatatatatg ctctctctcc 7260 ttggttccag cagcctcacc gatcccggccattttatagg cagtcataag caactgtgta 7320 gcacatctgc gaggtagaaa caggtaatacactgtgtagc acatctgagg tagaaacagc 7380 tgtggcagat ggatcacaac tggctgtattttaagctgca tgaggttcac gttgtggaac 7440 ccacttgggt tggatcgcgc gctgaagcccaatcacctct tccaactgct atttccgctc 7500 ggtgtcatca agacagagag agattcagagattctagaga acaacagtgc tcaaggaaaa 7560 caacccatac ccgctcgtta actgtcgtgttatctgtagt agtaaattca ggccgcgcac 7620 gtcgacggtt aactgttgtg ttatcctctttagtttagtt tgctacctaa cttgtcatac 7680 tttgtttaat tttttgtatc taaggttagttctttaattt ggatgactaa ttttagataa 7740 agtgtgacac agttagcgga atagtactatccggattttt ttttttggcg ccagtccggg 7800 caacaaatac aactgggctc tggcccccctatatacaaca aaattggctt tcaccaaaca 7860 acacgtcagc ccagacacat atttcattggcgtcattttc tttacacatt ataaaattga 7920 aacaaaaaga ctcacacttc agctgcttctgtcactctac ttcagaaaaa aaaatcatct 7980 atttttcttg atgcctgttt gatatataggtgtttatata tatagagaga gataatgagt 8040 ccctctcatc aagatatcca tatcataatggtacggtatt ttatggataa aaaagtacat 8100 cgtacgtatc acaaatctac aataatagaatggccatctc tgaaaacagt ttgatttagg 8160 tgcggtctgg tacaacaacg tcatagtatttttccatcag acaagtagat caacatttca 8220 tgcataagga atacgatggg taaaatatatttcattttag actaaatata catttattaa 8280 ttaacctatg aatatagttt gtatgtatatctatatttat tatcattcaa atgagtgtgg 8340 acggaaaaaa agaggctaga aagaactatatttcaaactg agggagtata aaagacacaa 8400 atttatacaa gtttcaagta taaagtacactacgtccagt gcagaaagcc gggatccccg 8460 ggcaggcctg caggtcgacg gccgagtactggcaggatat ataccgttgt aatt 8514 13 8692 DNA Artificial plasmid pTW018T-DNA sequence 13 cggcaggata tattcaattg taaatggctc catggcgatc gctctagaggatcttcccga 60 tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcgctatattttg 120 ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaacccatctcata 180 aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaacagaaattat 240 atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaactttattgccaaatg 300 tttgaacgat ctgcttcgga tcctagacgc gtgagatcag atctcggtgacgggcaggac 360 cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgtcatgccagtt 420 cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccgagcgcctcgtg 480 catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg accacgctcttgaagccctg 540 tgcctccagg gacttcagca ggtgggtgta gagcgtggag cccagtcccgtccgctggtg 600 gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgcgtgccttcca 660 ggggcccgcg taggcgatgc cggcgacctc gccgtccacc tcggcgacgagccagggata 720 gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcggctcggtacg 780 gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccgccggcatgtc 840 cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct gggtccatggttatagagag 900 agagatagat ttatagagag agactggtga tttcagcgtg tcctctccaaatgaaatgaa 960 cttccttata tagaggaagg gtcttgcgaa ggatagtggg attgtgcgtcatcccttacg 1020 tcagtggaga tgtcacatca atccacttgc tttgaagacg tggttggaacgtcttctttt 1080 tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggcagaggcatct 1140 tgaatgatag cctttccttt atcgcaatga tggcatttgt aggagccaccttccttttct 1200 actgtccttt cgatgaagtg acagatagct gggcaatgga atccgaggaggtttcccgaa 1260 attatccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgtatctttgaca 1320 tttttggagt agaccagagt gtcgtgctcc accatgttga cgaagattttcttcttgtca 1380 ttgagtcgta aaagactctg tatgaactgt tcgccagtct tcacggcgagttctgttaga 1440 tcctcgattt gaatcttaga ctccatgcat ggccttagat tcagtaggaactaccttttt 1500 agagactcca atctctatta cttgccttgg tttatgaagc aagccttgaatcgtccatac 1560 tggaatagta cttctgatct tgagaaatat gtctttctct gtgttcttgatgcaattagt 1620 cctgaatctt ttgactgcat ctttaacctt cttgggaagg tatttgatctcctggagatt 1680 gttactcggg tagatcgtct tgatgagacc tgctgcgtag gaacgcggccgcgtatacgt 1740 atcgatatct tcgaattcat atgcatgatc tggattttag tactggattttggttttagg 1800 aattagaaat tttattgata gaagtatttt acaaatacaa atacatactaagggtttctt 1860 atatgctcaa cacatgagcg aaaccctata ggaaccctaa ttcccttatctgggaactac 1920 tcacacatta ttatggagaa aatagagaga gatagatttg tagagagagactggtgattt 1980 cagcgtgtcc aagcttgcta gcctcagtcc acagcgaaga tcctcaccacagcggtcctg 2040 gtggagcccc acaggtcgtc cttgcaggac agggtgtaca ggaaggacctgtcggacttc 2100 acgttcttga agtccaggtt gatcctgtta gcgttcttgc cggaaccggagtccagcacg 2160 gtcttggtct gctcaccgat caccttgtag gtgaaggaga aggtacctggggtctgggta 2220 atcagtgggg agtcgatgaa gtaggtagcg tcgtggtact tcacggccagtgggtcaccg 2280 aagaagtcga agtcaccctc gatgatgtcg aactgtggca ctgggtccttgtactcgatc 2340 tcctcagcac ccacctccac gaaggacacg tcgtcccagt acacgttggtctggccgtca 2400 cccttgatgg agatggtgtt gatctcgttg ccctccaggt ttggcaccaggatgttgatc 2460 ctctggtaac ccacgtggtc cagggtgatg ttgtcggtca cgatggactcctgcttaccg 2520 tcgatctcga tgtccacgga caccttggag tcagccttca tgtacagggacacgtagtag 2580 tcgatgttct tcttcagctt gttcttggac tcggtggaca gctcggtgaaagcacccttg 2640 ttagcggacc tgtactgctt cttaccggtg ttaccaccgt tcaccacgtaggtgtagtac 2700 cagttgccga tcttggtgtt gtcggactca ccaccgtcgt acagggtggaggtctttatt 2760 gtgaagttca tcttcggttc gagcttcacg tcgtacaggt tcttcacgtccttgaaggaa 2820 ccggtggagt cgttcagctg cttcctgatc tgcttagcgg tgtactcgtccacgtaggac 2880 tgcacggaag cctcgaagat cggctggtcg ttgtagaaca gcaggccgtccttctcctcg 2940 atctcgtctg ggtaggccag cttcagggcc tccttcacgg ataaatttggggtcttgtcc 3000 tctgggttgg tgtagtcctt agcagccacc ctcttctcgg acatctggttgccgttgtcg 3060 acgatgatgc tggcggtctt ggcggagatc tcgtcggtga tgccgttccagtcaccagcg 3120 atggtgatgt tgccgttggt gtccttgatg gcgtacttgc cctccacctggtcggtttcg 3180 agcaggatcg gcttcttgtt ggacaggtag gtgttcagct gctccttgttcagtgggatc 3240 ggcctggagt tgaagtcgtc catcgtgttg atggcgatgc cgttcttgcccttctctggg 3300 taggactggt ctggcaggat ggtcagagcg gtggtgttct ccttggccttgatggtgccg 3360 atggtggtgc cgtccaggat gaaggaggtg gttggcttgg tctcgtagatagcaccggtg 3420 cccacgttgt tgtacctcac gttggcgttc aggtaggcgg actcagcaccgttgatgtgg 3480 gtgccgtcgt tggtggcgga gccccactcg ttggccacgg tctcggagtgctggtagttc 3540 acggacacgc cgaaggatgg acccagaccg gaccaaccag cgttcacgttcacgccctcg 3600 gtgttggtgt aggaccagtt ggtggactgg gaggactcca cggagtgggacaggtcctcg 3660 ttcttggaca ggatcacctt ttcgagggac acgttcacgg atgggaaagcagccaccagt 3720 gggttgaagg tctccttggc gttggacagt ggcatgtccc tagcagccttctcgtagtcg 3780 gagtatgggt cgcccacggt gtgggcttcg agtgggttgc tggtgaacttctggtagccc 3840 ttggaggcca gggagtcgtc ccacttcacg gccaccttgt tctggatggtgtagccgttc 3900 tcctcccaca cgtccgggat ggagtcgccg tcggtgtcgg tgtcctcgtcgatgtccctc 3960 ttggtcttct gggtgaacag gttggtcttg gaggccttct tcaggaacacctgggtctcc 4020 ttcttgttga actctgggtt cctcagctcg tcctgctgca cctgctgggagtggttctgg 4080 gagtcgatct tgaacagctt cagctccttg aagatcttgt tgtcgatgtgcagggcgtcg 4140 tcggactggt actcgatctt gatctgcacc agctggccct tttcgaggtgcacggactgc 4200 ttgttgttgc ccttctcgga gatcaccttg ccgtccagct cgatgatggcgttctcgtcg 4260 tcggacagct tgaaggtgaa gtcaccagtg gcggaggact ggatcaggccgatccagcgg 4320 atggagtggt actcctggtg cttctggtcc acgagggtgt tagcggtctgctggtcgtag 4380 atcagggtgt tgtccctggt tggagcgaac agggtcaggt cgttgaagtccttgcccttg 4440 aagtagtagc ccagcaggcc ctccctgtcg atctggttgt ccttcgaagcctgggtggtg 4500 gtggcgatag ccatggatcg actatgctct ctatctgatt ggtttggctttgctccagca 4560 gccagccatt ttataggcag cagtcactaa actgtaggct gtagcacgtctgacagacag 4620 gtagatggat cacaactggc tgtattttaa aaagctgcac gaggttcacgttgtgtcgtc 4680 gtggtataga taaatgtgca tgcagcaatg gaacaatatt ggggttgatgactgaatcgc 4740 tcaagctagc tagcccaatc atctcttcca actgctaccc gctgtgtctcataaacacgc 4800 aggtccagcg attctaaacc gcaacagtgc tcaacgaaaa ctacccttacccgctggtta 4860 attattgtgt tatcatattt aaatgctgtc attttcttta caaattataaaacttgggac 4920 gtgtttggtt cgctgcctat acttatttta tgtattggat tctatgcgcaaagagcaaaa 4980 ttccagtacc aaatgtttgt tgtattttat tgggtagcgt gtacgtcgcacattctgtaa 5040 tacaacctcc gttcacagat atatgacatg ttgatttttt taaaaaactttgaccattta 5100 tcttattcaa aagtataaaa ttttaattaa gcacaaacta ccttaagtgataaaacaaac 5160 cacacaaaaa ataaatgaca actcattatt ttttaaataa gacaagtgattaaagttttt 5220 taaaaagtca gcgatgtcat atatttatga acggtatata tatatatatatataacaccc 5280 atatcgagca ggcatcaaga aaaacatatc gatgattttt gttttcctaaagtagagtga 5340 caagctaaac aaatgacata tttttgtttc agttttgtaa ggccattctcagtggtgagc 5400 ttcagaacat gtgacatatt ttttttgttt tagttttgta aggcctttctcagtggtgag 5460 ctttagaaca agtgacatct ttttgtttca gttttgtaag gtcattcttagtggtgagct 5520 tcagaacaag tgagatgaga tctttttgtt tcagttttgt taggccagtttcatcggtga 5580 gcttcacaac aagtgacatc ttcttttcag ttttgtaagg ccattttcancggtgagctt 5640 cggtacaatg ttttccatgt tgtcacacca tatttaaact aggtaattgtatatatagaa 5700 ttttatctct atgaaactct accatctccc ataagctctt tctatatctctgcttttaat 5760 tgtatgtcat gtcactatgt atgatggtgt atcatcgtat ataatgagtatgaaattccg 5820 ccaatcacta ggggctaggc gcgccatatg catgatctgg attttagtactggattttgg 5880 ttttaggaat tagaaatttt attgatagaa gtattttaca aatacaaatacatactaagg 5940 gtttcttata tgctcaacac atgagcgaaa ccctatagga accctaattcccttatctgg 6000 gaactactca cacattatta tggagaaaat agagagagat agatttgtagagagagactg 6060 gtgatttcag cgtgtccaag cttgctagcc tcacttggtc agcagggtagcgtccactac 6120 gtacctcttc acacccttga tcaccacctc ggtgatcttg tcgatgtggtagttggagtc 6180 cttgtcgatc aggatctcct tctcggaagc gaagccaccg atagcggacaggtaggcacc 6240 agtggaaccc tttggcacct gcagcctcag gatgaacttc ctggagccgaaagcggacag 6300 cctctcggag gacagggagg tggacatgta acccttgtcc tccttcatggtgttcaggaa 6360 cttctcctcc atctccttca gggatggcag tgggtcggaa atctggtaaccgaactcggc 6420 cataccgcac cacctgtaca cggtgatgtt ctctgggatc ggctgcttctccagagcctc 6480 ggagatgttc ttgatctggg tgtccagctt ctcgttgccg gagccaccctggttccgcag 6540 gtagtcgttg atctgcttgt agtcctgcct agcgtagccg tccagggcctccctctgtgg 6600 gtcggtcagg ttcttagccc aaccctcgta gttcttcatg ccccacctgtgagcctcggc 6660 gttgatgtcg ttcttgaagt ccagggactt cttcagggta ccctggatctgcaggcactc 6720 gtaacccttc ttcaccacct tggagatgtt gtccacgtgc agcacgtagccgttgtcgat 6780 cagcatcttg tactcgttgt tgttcaggat cacaccagcc ttggtcgggatggtggagcc 6840 cttgccggat ggcacggtca cttggaggat gatcctctcc ttggaggacacgttctgagc 6900 agtcaggtgg gtgtccaggt aggagtcgaa cttgatgtcc ttgcccaggaactgctcctt 6960 gaactgggcc tgcacgtcgg tgttgatggt gttgccctcg gtcagtggcttgttaaatcc 7020 gatggtggat ggctccacgt tcttgtaggt cacgatggag gaggacaggttggccttgtc 7080 gaacatcttg tcgatctcct tcaggtcctt gatctcgtcc tcgaaggagccagccatgct 7140 gaaggtgatc tccttgtagt tcttcttgat gtcgttcttg ttgtccaggaaattattcat 7200 acgcgtcttc tcggtcacgg tcagcttcca ctccttctcc ttctccttgccccactcctt 7260 ggccttctcc ttgtcctcct tgaagtcctc ggtgttgttg gtggtcttcactagtgccat 7320 ggatcgacta tgctctctat ctgattggtt tggctttgct ccagcagccagccattttat 7380 aggcagcagt cactaaactg taggctgtag cacgtctgac agacaggtagatggatcaca 7440 actggctgta ttttaaaaag ctgcacgagg ttcacgttgt gtcgtcgtggtatagataaa 7500 tgtgcatgca gcaatggaac aatattgggg ttgatgactg aatcgctcaagctagctagc 7560 ccaatcatct cttccaactg ctacccgctg tgtctcataa acacgcaggtccagcgattc 7620 taaaccgcaa cagtgctcaa cgaaaactac ccttacccgc tggttaattattgtgttatc 7680 atatttaaat gctgtcattt tctttacaaa ttataaaact tgggacgtgtttggttcgct 7740 gcctatactt attttatgta ttggattcta tgcgcaaaga gcaaaattccagtaccaaat 7800 gtttgttgta ttttattggg tagcgtgtac gtcgcacatt ctgtaatacaacctccgttc 7860 acagatatat gacatgttga tttttttaaa aaactttgac catttatcttattcaaaagt 7920 ataaaatttt aattaagcac aaactacctt aagtgataaa acaaaccacacaaaaaataa 7980 atgacaactc attatttttt aaataagaca agtgattaaa gttttttaaaaagtcagcga 8040 tgtcatatat ttatgaacgg tatatatata tatatatata acacccatatcgagcaggca 8100 tcaagaaaaa catatcgatg atttttgttt tcctaaagta gagtgacaagctaaacaaat 8160 gacatatttt tgtttcagtt ttgtaaggcc attctcagtg gtgagcttcagaacatgtga 8220 catatttttt ttgttttagt tttgtaaggc ctttctcagt ggtgagctttagaacaagtg 8280 acatcttttt gtttcagttt tgtaaggtca ttcttagtgg tgagcttcagaacaagtgag 8340 atgagatctt tttgtttcag ttttgttagg ccagtttcat cggtgagcttcacaacaagt 8400 gacatcttct tttcagtttt gtaaggccat tttcancggt gagcttcggtacaatgtttt 8460 ccatgttgtc acaccatatt taaactaggt aattgtatat atagaattttatctctatga 8520 aactctacca tctcccataa gctctttcta tatctctgct tttaattgtatgtcatgtca 8580 ctatgtatga tggtgtatca tcgtatataa tgagtatgaa attccgccaatcactagggg 8640 caggcctgca ggtcgacggc cgagtactgg caggatatat accgttgtaatt 8692 14 500 DNA Zea mays misc_feature (127)..(127) n=any nucleotide14 ggcggaattt catactcatt atatacgatg atacaccatc atacatagtg acatgacata 60caattaaaag cagagatata gaaagagctt atgggagatg gtagagtttc atagagataa 120aattctntat ntacaattac ctagtttaaa tatggngtga caacatggaa aacattgtac 180cgaagctcac cgntgaaaat ggccttacaa aactgaaaag aagatgtcac ttgttgtgaa 240gctcaccgat gaaactggcc taacaaaact gaaacaaaaa gatctcatnt cacttgttct 300gaagctcacc actaagaatg accttacaaa actgaaacaa aaaagatgtc cttgttctaa 360agctcaccac tgagaaaggc cttacaaaac taaaacaaaa aaaatttgtc acatgtcttg 420aagctcacca ctgagaatgg ccttacaaaa ctgaacaaaa atntgtcatt tggtttagct 480ttgtcnctct actttaagga 500 15 604 DNA Zea mays 15 tgaataagat aaatggtcaaagttttttaa aaaaatcaaa catgtcatat atctgtgaac 60 ggaggttgta ttacagaatgtgcgacgtac acgctaccca ataaaataca acaaacattt 120 ggtactggaa ttttgctctttgcgcataga atccaataca taaaataagt ataggcagcg 180 aaccaaacac gtcccaagttttataatttg taaagaaaat gacagcattt aaatatgata 240 acacaataat taaccagcgggtaagggtag ttttcgttga gcactgttgc ggtttagaat 300 cgctggacct gcgtgtttatgagacacagc gggtagcagt tggaagagat gattgggcta 360 gctagcttga gcgattcagtcatcaacccc aatattgttc cattgctgca tgcacattta 420 tctataccac gacgacacaacgtgaacctc gtgcagcttt ttaaaataca gccagttgtg 480 atccatctac ctgtctgtcagacgtgctac agcctacagt ttagtgactg ctgcctataa 540 aatggctggc tgctggagcaaagccaaacc aatcagatag agagcatagt cgatccatgg 600 catg 604 16 25 DNAArtificial oligonucleotide primer GVK22 16 gctgtgtgag agtagtagtg gcttc25 17 24 DNA Artificial oligonucleotide primer GVK23 17 cacaagcgtggctgacagca tcgt 24 18 26 DNA Artificial oligonucleotide primer GVK24 18gctcacgaga ggcagcgcgc gtcgtc 26 19 21 DNA Artificial oligonucleotideprimer GVK25 19 gtaaaagttg tggcttcccg g 21 20 25 DNA Artificialoligonucleotide primer GVK26 20 ccgcgctgtg cccgacagct taaac 25 21 27 DNAArtificial oligonucleotide primer GVK31 21 gggttggatc gcgccaatca cctcttc27 22 24 DNA Artificial oligonucleotide primer GVK32 22 ccgctcggtgtcatcaagac agag 24 23 26 DNA Artificial oligonucleotide primer GVK33 23gctcaaggaa aacaacccat acccgc 26 24 24 DNA Artificial oligonucleotideprimer GVK38 24 ggcgccagtc cgggcaacaa atac 24 25 25 DNA Artificialoligonucleotide primer GVK39 25 gggctctggc ccccctatat acaac 25 26 27 DNAArtificial oligonucleotide primer GVK45 26 cgggatcccg gctttctgca ctggacg27 27 15 DNA Artificial oligonucleotide primer MDB285 27 ntcgastwtsgwgtt 15 28 16 DNA Artificial oligonucleotide primer MDB286 28ngtcgaswga nawgaa 16 29 16 DNA Artificial oligonucleotide primer MDB36329 csggntgawn taawac 16 30 15 DNA Artificial oligonucleotide primerMDB364 30 sscgnaawtt catwc 15 31 15 DNA Artificial oligonucleotideprimer MDB552 31 ngtcsagwaw scatt 15 32 16 DNA Artificialoligonucleotide primer MDB556 32 cngasnagwt wgcata 16

What is claimed is: 1) A corn root preferential promoter fragment comprising a nucleotide sequence selected from the following group of nucleotide sequences: a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 1 to the nucleotide at position 338 or SEQ ID No 2 from the nucleotide sequence at position 11 to the nucleotide at position 1196; b) a nucleotide sequence comprising the nucleotide sequence of SEQ ID No 15 from the nucleotide at position 1 to the nucleotide at position 1280; and c) a nucleotide sequence comprising the nucleotide sequence of an about 400 bp to an about 1300 bp DNA fragment hybridizing under stringent conditions with a DNA fragment having said nucleotide sequence mentioned under a) or b). 2) A corn root preferential promoter region comprising a corn root preferential promoter according to claim
 1. 3) The corn root preferential promoter region according to claim 2, further comprising the nucleotide sequence of SEQ ID 1 from the nucleotide at position 339 to the nucleotide at position
 366. 4) The corn root preferential promoter region according to claim 2, further comprising the nucleotide sequence of SEQ ID 14 from the nucleotide at position 1281 to the nucleotide at position
 1308. 5) A chimeric gene comprising the following operably linked DNA regions a) a corn root preferential promoter according to claim 1; b) a heterologous DNA region encoding a biologically active RNA of interest; and c) a transcription termination and polyadenylation signal. 6) The chimeric gene according to claim 5, wherein said biologically active RNA encodes a protein of interest. 7) The chimeric gene according to claim 6, wherein said protein is a protein which when expressed in the cells of a plant confers pest or pathogen resistance to said plant. 8) The chimeric gene according to claim 7, wherein said protein is ISPA1 or ISPA2 from Brevibacillus laterosporus. 9) A plant cell comprising a chimeric gene according to any one of claims 5 to
 8. 10) A plant comprising in its cells a chimeric gene according to any of claims 5 to
 8. 11) The plant according to claim 10, which is a corn plant. 12) A seed of a plant comprising in its cells a chimeric gene according to any one of claims 5 to
 8. 13) A method for expressing a biologically active RNA preferentially in the roots of a plant, said method comprising a) providing the cells of the roots of said plants with a chimeric gene according to any one of claims 5 to 8; and b) growing said plants. 14) The method according to claim 13, wherein said plant is a corn plant. 15) An isolated DNA molecule comprising a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID No 4 or SEQ ID No
 6. 16) An isolated DNA molecule comprising a nucleotide sequence selected from the group of SEQ ID No 3; SEQ ID No 5 and SEQ ID No
 11. 17) A method for isolating a corn root preferential promoter region, comprising the steps of: a) identifying a genomic fragment encoding an RNA transcript from which a cDNA can be synthesized, said cDNA comprising the nucleotide sequence of SEQ ID 3 or SEQ ID No 5 or functional equivalents; b) isolating a DNA region upstream of a nucleotide sequence encoding the protein with the amino acid of SEQ ID No 4 or SEQ ID No 6 or functional equivalents. 18) A corn root preferential promoter obtained by the method of claim
 17. 